PROJECT SUMMARY/ABSTRACT Seasonal influenza A virus (IAV) infections account for over 700,000 hospitalizations and 50,000 annual deaths in the US alone. Moreover, highly virulent H5 and H7 strains of avian IAV, while currently limited in their spread between humans, are only a few mutations from acquiring the capacity for widespread transmissibility. As current vaccines and antiviral strategies are either limited in their efficacy or susceptible to viral resistance and evasion, identifying new therapeutic entry-points for seasonal and virulent IAV disease, preferably those that target pathogenic host signaling pathways, is an urgent imperative. We have identified the host kinase RIPK3 as a promising new entry point for therapeutic development against IAV. RIPK3 is the central mediator of a highly pro-inflammatory form of cell death termed necroptosis, which we have found is a major contributor to lung injury and inflammation during IAV infection. Both seasonal and pandemic strains of IAV trigger RIPK3- dependent necrotic lung damage that we propose underlies Acute Respiratory Distress Syndrome (ARDS), as well as viral and bacterial pneumonia, each of which remain major causes of morbidity and mortality following IAV infection. Notably, RIPK3 also mediates or amplifies a range of chronic TNF-?-mediated pathologies (such as rheumatoid arthritis) making it a very attractive new molecular target for multiple inflammatory conditions. Curiously, given how important a therapeutic target RIPK3 potentially is, no selective RIPK3 inhibitors have been advanced into clinical trials. We now have developed a new structural class of RIPK3 inhibitor, which we call the UH15 series, and which is based on a pyrido[2,3-d]pyrimidine scaffold that targets both the ATP- as well as the allosteric Glu-out pockets of RIPK3. Our preliminary findings reveal that UH15 analogs, after just one round of optimization, are already more potent than current RIPK3 inhibitors and display promising activity against IAV induced necrosis in vitro and in vivo. These exciting results highlight the immediate translational potential of the UH15 series for necrotic lung injury and consequent ARDS and pneumonia triggered by seasonal and virulent strains of IAV. The goals of our proposal are to iteratively optimize UH15-based compounds for RIPK3 blockade in vitro and, by use of a rapid mouse model of RIPK3-mediated pathology (the TNF SIRS model), prioritize compounds for use in vivo (Aim 1). We then propose to assess these UH15 compounds for therapeutic efficacy in a variety of IAV-triggered disease settings, including the scenarios of (1) high-risk seasonal IAV infections, (2) infection by highly pathogenic avian IAV, and (3) secondary pneumococcal pneumonia following seasonal IAV infection (Aim 2). The proposed studies bring together a team of researchers with strong, complementary expertise in small-molecule medicinal chemistry (Cuny), RIPK3 kinase biochemistry and function in inflammation (Degterev), and RIPK3-mediated cell death signaling during IAV pathogenesis (Balachandran, Thomas). Successful completion of these Aims has the potential to transform the treatment of multiple IAV-induced diseases initiated or amplified by necrotic lung injury.